Hepatitis B virus (HBV) is the most significant of the hepatotrophic viruses in terms of the number of people chronically infected and the severity of the complications of infection. It is a major cause of human liver disease which can lead to chronic infection, cirrhosis and hepatocellular carcinoma, resulting in over a million deaths worldwide each year.
HBV is a double stranded DNA virus that may be carried by as much as 20% or more of the apparently healthy population in certain parts of the world, such as Africa, Asia and the Pacific Region (Principles and Practice of Clinical Virology, 3rd Edition, Chapter 2: Hepatitis Viruses, pp. 162-180). The reservoir of carriers worldwide is estimated at a number over 300 million. HBV was originally thought to be spread exclusively by blood and blood products, although it now appears that HBV can also be transmitted by intimate contact, such as sexual contact, and other routes may also be possible. Thus, transmission of infection may result from accidental inoculation of minute amounts of blood, or fluid contaminated with blood, during medical, surgical and dental procedures; immunization with inadequately sterilized syringes and needles; intravenous or percutaneous drug abuse; tatooing; ear, nose and other piercing; acupuncture; laboratory accidents; and, accidental inoculation with razors and similar objects that have been contaminated with blood.
The genomes of a variety of isolates of HBV have been cloned and the complete nucleotide sequence thereof determined. Although there is some variation in sequence (up to about 12% of nucleotides) between these isolates, the genetic organization and other essential features are conserved. The genome is around 3200 base pairs in length and analysis of the protein coding potential reveals four conserved ORFs. The four ORFs are located on the same DNA strand and the strands of the genome have accordingly been designated the plus (incomplete strand) and minus (complete strand).
HBV belongs to the hepadnaviridae family and consists of an outer envelope of host-derived lipids containing a virion-encoded surface antigen (HBsAg). This 42 nm lipoprotein shell encloses an icosahedral nucleocapsid assembled from the core antigen (HBcAg) that contains the viral genomic DNA and the viral polymerase (for review, see Nassal and Schaller, 1993). The core protein is the cytoplasmic product of the C-gene, composed of 183 or 185 amino acid residues (21 kDa) depending on sero-subtypes, which can be divided into an N-terminal assembly domain (residues 1-149) and a C-terminal very basic protamine-like domain (residues 150-185), respectively responsible for polymerization into particles and RNA packaging. The HBc protein has the ability to form disulfide-linked homodimers which spontaneously assemble into particles (Zhou and Standring, 1992).
HBcAg is a very powerful immunogen inducing strong humoral, T helper (Th) and cytotoxic T cell (CTL) responses and functioning as both T-cell-dependent and T-cell-independent antigen (Milich et al., 1997a, b). Anti-HBc arise in virtually all infected individuals. They are produced very early after infection and may be detected a few days after the detection of HBsAg in the blood of infected subjects. Moreover, in acute infections, HBsAg declines over a period of several weeks and is replaced by detectable levels of HBsAg antibody (anti-HBs). During a xe2x80x9cwindowxe2x80x9d period, when neither HBsAg nor its homologous antibody is detectable, anti-HBc may be the only detectable serological marker of HBV infection. In addition, anti-HBc usually persists longer than any other HBV marker. Anti-HBc is therefore the most useful marker for the diagnosis of an ongoing or past HBV infection and for epidemiological purposes.
Intensive studies have shown that HBcAg can be produced in a variety of heterologous expression systems including E. coli and undergoes correct folding and self-assembly to form core particles similar to native capsids (Pasek et al., 1979; Cohen and Richmond, 1982; Naito et al., 1997; Wizeman and von Brunn, 1999). Bacterially expressed HBc molecules assemble into particles of two sizes arranged respectively with a triangulation number T=3 (90 dimers) or T=4 (120 dimers) icosahedral symmetry (Crowther et al., 1994; Wingfield et al., 1995). The physiological implications of this dimorphic switch are not clear, although the T=4 form is reported to outnumber the T=3 form by xcx9c13 to 1 in capsids isolated from the human liver (Kenney et al., 1995). The crystal structure of the T=4 capsid of the bacterially expressed truncated protein (aa .1-149) has been solved by X-ray crystallography to 3.3 xc3x85 resolution (Wynne et al., 1999). The monomer fold is characterized by four xcex1-helices and the absence of xcex2-sheets. In agreement with previous biochemical analyses, the structural data revealed two regions required for the dimerization of core monomers and for the subsequent assembly of the dimers into core particles (Wynne et al., 1999).
Many different procedures have been described to purify HBcAg. Common purification procedures are based on sedimentation of the core particles on sucrose gradients which do not allow the removal of all contaminating E. coli material and are often associated with a low yield. As a consequence, most of the commercially available anti-HBc detection systems are immunoassays based on inhibition or competition where human anti-HBc inhibit a labeled anti-HBc from binding to an immobilized recombinant HBcAg (Pujol et al., 1994). Consequently, the presence of anti-HBc in the sample generates a low signal value, whereas its absence results in a high signal value. This conventional test format circumvents the need for highly purified HBcAg, but has potential drawbacks which include poor specificity and poor reproducibility especially near assay cutoff (Dodd and Popovsky, 1991). False-positive anti-HBc reactivity has been attributed to cross-reactive antibodies or interfering substances in human serum (Robertson et al., 1991). It has been shown that the pretreatment of serum samples with reducing agents could significantly improve the specificity of anti-HBc determination in competitive assay (Robertson et al., 1991; Spronk et al., 1991; Weare et al., 1991). However in order to avoid cost-intensive remeasurements in routine diagnosis and discarding of blood donations due to (false)-positive anti-HBc results, there is still a need for increasing the test""s specificity.
In an attempt to improve the production of a high-quality, highly purified recombinant antigen for use in diagnosis of anti-HBc antibodies in biological samples, HBcAg was expressed in yeast. The highly purified recombinant protein was characterized and its suitability as a diagnostic antigen was evaluated, by way of example, in a new sandwich enzyme immunoassay (EIA) in which anti-HBc antibodies were captured by binding to recombinant HBcAg on a solid phase and then detected by using recombinant HBcAg labeled with an appropriate marker.
As explained hereabove, assays for the diagnosis of the potential presence of HBV in a patient, as indicated by the presence of anti-HBc antibodies, are known in the art. However, it is believed that such assays are not sufficiently sensitive and specific and a need exists for the production of a high-quality recombinant HBcAg allowing the development of reliable tests, in particular sandwich assays.
Consequently, the subject of the present invention is a new expression system or cassette which is functional in a cell derived from a yeast selected from the group consisting of strain Pichia and Schizosaccharomyces, especially selected from the group consisting of Pichia pastoris, Pichia methanolica and Schizosaccharomyces pombe and allowing the expression of HBc DNA or fragments thereof encoding HBcAg or fragments thereof, placed under the control of the elements necessary for its expression. A large number of these cells are commercially available in collections such as ATCC (Rockville, Md., USA) and AFRC (Agriculture and Food Research Council, Norfolk, UK). For the purpose of the present invention, said cell may be of the wild type or mutant type. The expression cassette according to the invention comprises elements necessary for the expression of HBcAg or fragments thereof in the considered cells.  less than  less than Components necessary for the expression greater than  greater than  is understood to mean all the elements which allow the transcription of a DNA or DNA fragment into mRNA and the translation of the latter into protein. Among these, the promoter region is of special importance. The promoter region may be autologue, i.e, the promoter region of the C-gene of HBV or heterologue, i.e, the promoter region may corresponds to a promoter of a plasmid or promoter of a gene in the host cell. It can be constitutive, that is to say it can allow a constant transcription level during the entire cell cycle. However, it may be advantageous to use a regulable promoter region which makes it possible to vary the transcription levels according to the culture conditions or the cell growth phase depending on the presence of an inducer (activation of transcription) or of a repressor (repression). Generally, the regulable promoter regions are derived from regulable genes for which the regulation mechanisms may be highly varied. On the other hand, an expression cassette according to the invention may, in addition, contain other elements which contribue to the expression of the DNA or fragments thereof as well as transcription activation sequences.
The present invention is also directed to a vector comprising an expression cassette of the present invention. It may be a replicating plasmid vector before integration of DNA or fragments thereof It may be a multicopy vector present between about 1 and 500 copies, preferably between 1 and 20 copies, in the host cell. There may be mentioned, by way of exemple, the vectors derived from pPIC3.5K (Invitrogen).
The present invention also relates to a yeast cell selected from the group consisting of cells of strains Pichia and Schizosaccharomyces, especially selected from the group consisting of Pichia pastoris, Pichia methanolica and Schizosaccharomyces pombe, and comprising an expression cassette of the invention either in a form integrated into the cell genome or inserted into a vector.
The present invention also embraces the recombinant HBcAg or fragments thereof produced by an expression cassette, a vector or a cell according to the invention. Within the framework of the present invention, HBcAg or fragments thereof may be modified in vitro, especially by addition or deletion of chemical groups, such as phosphates, sugars or myristic acid so as to enhance its stability or the presentation of one or more epitope(s).
The present invention also relates to use of the recombinant HBcAg or fragments thereof of the present invention in diagnosis of anti-HBc antibodies in samples to be tested. With respect to recombinant HBcAg expressed in procaryote cells, such as E. coli cells, the recombinant protein or fragments of the present invention is particularly interesting because it is produced in a highly purified form and does not need additional and time consuming steps of purification because the protein is folded, that means close to the native form. Consequently, a test using the protein of the present invention in diagnosis of anti-HBc antibodies in a sample to be tested is more reliable, more specific and more sensitive. The protein of the present invention is particularly attractive in the sandwich method. In the following examples, comparison is made between HBcAg expressed in E. coli cells and HBcAg expressed in Pichia, when used in diagnostic test.
Thus the present invention relates to a reagent for the detection and/or the monitoring of an HBV infection, which comprises, as reactive substance the recombinant protein or fragments thereof of the present invention. The above reagent is or will be attached directly or indirectly onto an appropriate support. The support may be, without limitation, in the form of a cone, a tube, a well, beads or the like. The term  less than  less than solid support greater than  greater than  as used here includes all materials on which a reagent may be immobilized for use in diagnostic tests. Natural or synthetic materials, chemically modified or otherwise, can be used as solid support, especially polysaccharides such as cellulose materials, cellulose derivatives; polymers such as polystyrene, polyacrylate, polyethylene, vinyl chloride or copolymers such as propylene and vinyl chloride polymer, vinyl chloride and vinyl acetate polymer; styrene-based copolymers; natural fibers and synthetic fibers. Preferably, the support is a polystyrene polymer or a butadiene-styrene copolymer. The attachment of the reagent onto the solid support may be performed in a direct or indirect manner. All techniques for the attachment of a reagent onto a solid support are well known.
The present invention also relates to a process for the detection of anti-HBc antibodies in a biological sample to be tested, such as blood, plasma or serum, from an individual or from an animal likely to be or to have been infected by HBV according to which at least the following steps are performed:
a mixture is prepared comprising:
(i) a reagent as defined above which is or which will be immobilized on a solid support,
(ii) the sample to be tested comprising if they are present anti-HBc antibodies,
(iii) a labeled ligand which will be capable to react with the sample anti-HBc antibodies;
the mixture is incubated for a predetermined time;
the solid phase is separated from the liquid phase; and
the possible presence of anti-HBc antibodies is revealed by measuring the level of labeling in the solid phase.
This process named  less than  less than sandwich assay greater than  greater than  may be performed in one or further step(s) and the ligand is either a labeled recombinant HBcAg of the present invention or a labeled anti-immunoglobulin.
The present invention embraces another process for the detection of anti-HBc antibodies in a biological sample to be tested, such as blood, plasma or serum, from an individual or from an animal likely to be or to have been infected by HBV according to which at least the following steps are performed:
a mixture is prepared comprising:
(i) a reagent as defined above which is or which will be immobilized on a solid support,
(ii) the sample to be tested comprising if they are present anti-HBc antibodies,
(iii) labeled anti-HBc antibodies which will be capable to react with the reagent;
the mixture is incubated for a predetermined time;
the solid phase is separated from the liquid phase; and
the possible presence of anti-HBc antibodies is revealed by measuring the level of labeling in the solid phase.
This process is named  less than  less than competition assay greater than  greater than .
Because HBcAg is a very powerful immunogen inducing strong humoral response, the recombinant HBcAg of the present invention is used for the production of monoclonal or polyclonal antibodies or fragments thereof obtained by the immunological reaction of an animal organism, preferably a mouse, a rat or a rabbit to an immunogenic agent consisting of the recombinant HBcAg or fragments thereof of the present invention. The production of polyclonal and monoclonal antibodies forms part of the general knowledge of persons skilled in the art. By way of reference, mention may be made of Kxc3x6hler G. and Milstein C. (1975) and Galfre G. et al. (1977) for the production of monoclonal antibodies and Roda A., Bolelli G. F. (1980), for the production of polyclonal antibodies. Antibodies can also be produced by immunizing mice, rat or rabbit with the HBcAg or fragments thereof of the present invention. For the production of monoclonal antibodies, the immunogene can be coupled to Keyhole Limpet haemocyanin (KLH peptide) as a support for the immunization, or to serum albumin (SA peptide). The animals are subjected to an injection of immunogene using complete Freund""s adjuvant. The sera and the hybridoma culture supernatants derived from the immunized animals are analysed for their specificity and their selectivity, using conventional techniques such as, for example, ELISA assays or Western Blot. The hybridomes producing the most specific and the most sensitive antibodies are selected. Monoclonal antibodies can also be produced in vitro by cell culture of the hybridomes produced or by recovering ascites fluid after intraperitoneal injection of the hybridomes into mice. Whatever the method of production, by supernatant or by ascites, the antibodies are then purified. The purification methods used are essentially ion exchange gel filtration and exclusion chromatography or affinity chromatography (protein A or G). A sufficient number of antibodies are screened in functional assays in order to identify the most effective antibodies. The in vitro production of antibodies, of antibody fragments or of antibody derivatives, such as chimeric antibodies produced by genetic engineering, is well known to persons skilled in the art.
More particularly, the term xe2x80x9cantibody fragmentxe2x80x9d is intended to mean F(ab)2, Fab, Fabxe2x80x2 and sFv fragments (Blazar et al., (1997) and Bird et al., (1988,)) of a native antibody, and the term xe2x80x9cderivativexe2x80x9d is intended to mean, inter alia, a chimeric derivative of a native antibody (see, for example, Arakawa et al., (1966) and Chaudray et al., (1989)).
The monoclonal or polyclonal antibody thus obtained is incorporated into a diagnostic composition which is used in a method for detecting at least the HBc protein in a biological sample after pretreatment, if necessary, of the sample to disrupt the cells and release the HBc protein, according to which the biological sample is brought into contact with said diagnostic composition under predetermined conditions which allow the formation of antibody/antigen complexes, and the formation of said complexes is detected. Said composition can also comprise at least one monoclonal or polyclonal antibody or the fragments thereof, directed against HBsAg protein.
The present invention also relates to a sandwich or competition process for detecting at least the HBc protein in a biological sample, if necessary after pretreatment of the sample, to disrupt the cells and release the HBc protein, said processes preferably using at least one monoclonal antibody of the invention.
According to the present invention the sandwich process comprises at least the following steps:
a mixture is prepared comprising:
(i) a reagent which consists of at least one monoclonal antibody of the invention which is or which will be immobilized on a solid support,
(ii) the sample to be tested comprising if it is present HBcAg,
(iii) a labeled ligand which will be capable to react with the sample HBcAg;
the mixture is incubated for a predetermined time;
the solid phase is separated from the liquid phase; and
the possible presence of HBcAg is revealed by measuring the level of labeling in the solid phase. The labeled ligand may be a monoclonal or polyclonal antibody.
In another embodiment of the present invention the sandwich process comprises at least the following steps:
a mixture is prepared comprising:
(i) a reagent which consists of at least one polyclonal antibody of the invention which is or which will be immobilized on a solid support,
(ii) the sample to be tested comprising if it is present HBcAg,
(iii) a labeled ligand which will be capable to react with the sample HBcAg;
the mixture is incubated for a predetermined time;
the solid phase is separated from the liquid phase; and
the possible presence of HBcAg is revealed by measuring the level of labeling in the solid phase. The labeled ligand may be a monoclonal or polyclonal antibody.
According to the present invention the competition process comprises at least the following steps:
a mixture is prepared comprising:
(i) a reagent which consists of at least one monoclonal antibody or polyclonal antibody of the invention which is or which will be immobilized on a solid support,
(ii) the sample to be tested comprising if it is present HBcAg,
(iii) a labeled ligand which will be capable to react with the reagent (i) and which consists of a recombinant HBcAg of the invention;
the mixture is incubated for a predetermined time;
the solid phase is separated from the liquid phase; and
the possible presence of HBcAg is revealed by measuring the level of labeling in the solid phase.
Because HBcAg is a very powerful immunogen inducing strong humoral response the recombinant HBcAg of the invention is usable as an active component of the immune response. Thus, a subject of the present invention is also a vaccine against the HBV virus. This vaccine is prepared according to already known methods used for preparing commercially available vaccines. This vaccine comprises at least the recombinant HBcAg protein of the invention or fragments thereof The recombinant HBcAg protein is obtained by using an expression cassette of the present invention.
An immunogenic or vaccine composition according to the invention is a composition which comprises a recombinant HBcAg protein or a protein fragment as defined above, optionally combined with a suitable vehicle and/or adjuvant and/or a pharmaceutically acceptable excipient. The vaccines comprising the HBcAg protein of the invention, or the fragments thereof, are prepared conventionally and contain an immunoprotective amount of the HBcAg protein, or of the fragments thereof, preferably in a buffered saline solution and mixed or adsorbed using known adjuvants such as aluminium hydroxide or phosphate.
The term xe2x80x9cimmunoprotectivexe2x80x9d means that an amount of the recombinant HBcAg protein of the invention or of the fragments thereof which is sufficient to induce a production of antibodies (humoral immune response) which is sufficient to be protective, or a cytotoxic cell-mediated immune response (cellular immune response), to confer a protection against the infectious agent without inducing side effects, is administered to an individual. The two types of response differ in that the antibodies recognize the antigens in their three-dimensional form, whereas the cytotoxic cells recognize portions of said antigens, associated with glycoproteins encoded by the major histocompatibility complex (MHC). Cytotoxic T lymphocytes (CTLs) play an essential role in the defence of cells infected with viruses. They act directly by cytotoxicity, but also by providing specific and nonspecific help to other hematopoietic cells, such as macrophages, B cells and other T cells. The infected cells transform the antigen through intracellular events involving proteases. The transformed antigen is then presented at the surface of the cells, in the form of peptides bound to class I HLA molecules, to the T-cell receptors on the CTLs. Class I MHC molecules can also bind exogenous peptides and present them to CTLs without intracellular transformation. Chisari et al., (Microbiol. Pathogen. 6:31 (1989)) have suggested that the liver lesions could be mediated by a class I HLA-restricted CD8+cytotoxic T-cell response to the antigens encoded by HBV.
The amount of HBcAg protein of the invention or fragments thereof depends on whether adjuvant is added or not, but generally is between 10 and 50 xcexcg/ml of protein or of fragment. Thus, commonly, 20 xcexcg/0.5 ml of protein in adults and 10 xcexcg/0.5 ml in children are administered per dose. The vaccines can also comprise other proteins which enhance the immune response. The HBcAg interacts with certain enhancing proteins, preferably an albumine and/or an unprocessed structural protein from a positive stranded RNA virus, to provide a complex comprised of the HBcAg and the albumin or unprocessed structural protein from the positive stranded RNA virus. Pursuant to such complexing, the HBcAg is believed to undergo conformational changes that enhance the antigenicity of the HBcAg when compared to HBcAg alone in terms of both or either affinity or specificity. The HBcAg protein of the invention, or the fragments thereof, can also be mixed with HBsAg and/or Pre-S proteins or fragments of said proteins, for the formulation of a vaccine. They can also be mixed with hybrid particles carrying epitopes of proteins of other organisms, or with other immunogenic compounds, for the formulation of bivalent or multivalent vaccines. The preparation of vaccines is in particular described in xe2x80x9cVaccinesxe2x80x9d, ed. Voller et al., University Park Press, Baltimore, Md., USA, 1978.
The vaccine is administered at a given dose in one or more intramuscular or subcutaneous injections, followed by (a) booster(s), if necessary. The immunizing effect of the vaccine is monitored by assaying anti-HBc antibodies in the vaccinated individual.
The administration of (a) derived protein(s) or peptide(s) of interest, or the fragment(s) thereof, alone or in combination is used for prophylaxis and/or therapy. These administered proteins or peptides are characterised in that they do not exhibit the virulence of HBV, but are capable of inducing a humoral or cellular immune response in the individual to whom they are administered. Such proteins are termed xe2x80x9cmodifiedxe2x80x9d, but their immunogenicity is conserved.
The identification of (a) vaccine protein(s) or fragment(s) is carried out as follows:
the xe2x80x9cmodifiedxe2x80x9d candidate molecules are analysed in a functional assay to be sure that they have lost their toxicity and to verify their immunogenicity, (i) by carrying out an in vitro assay of proliferation of CD4+T lymphocytes specific for the antigen administered (T cell assay) or an in vitro assay of cytotoxicity of the CD8+lymphocytes specific for the antigen administered, and (ii) by measuring, inter alia, the level of circulating antibodies directed against the natural protein. These modified forms are used to immunize humans using standardized procedures with suitable adjuvants.
The prepared vaccines are injectable, i.e. in liquid solution or in suspension. As an option, the preparation can also be emulsified. The antigenic molecule can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Examples of favorable excipients are water, a saline solution, dextrose, glycerol, ethanol or equivalents, and combinations thereof If desired, the vaccine can contain minor amounts of auxiliary substances such as wetting agents or emulsifiers, agents which buffer pH or adjuvants such as aluminium hydroxide, muramyl dipeptide or variations thereof In the case of the peptides, their coupling to a larger molecule (KLH, tetanus toxin) sometimes increases immunogenicity. The vaccines are conventionally administered by injection, for example intramuscular injection.
The expression xe2x80x9cpharmaceutically acceptable vehiclexe2x80x9d is intended to mean the supports and vehicles which can be administered to humans or to animals, as described, for example, in Remington""s Pharmaceutical Sciences 16th ed., Mack Publishing Co. The pharmaceutically acceptable vehicle is preferably isotonic, hypotonic or is weakly hypertonic with a relatively low ionic strength. The definitions of the pharmaceutically acceptable adjuvants and excipients are also given in Remington""s Pharmaceutical Sciences mentioned above.
The present invention also relates to a pharmaceutical composition intended for the treatment or for the prevention of a HBV infection in an individual or animals, comprising a therapeutically effective quantity of the expression cassette, the vector or the cell as defined above.
Finally, the present invention concerns a process for the vaccination of an individual (human or animal) according to an active immunotherapeutic composition, especially a vaccinal preparation, as defined above is injected to the individual; and a process for the treatment or for the prevention of a HBV infection in which a pharmaceutical composition as defined above is administrated to the individual (human or animal).
The peptide sequence of FIG. 1A corresponds to a sequence subtype adw2, but of course the expression cassette according the invention allows the expression of any genotype or subtype.
The examples below will make it possible to demonstrate characteristics and advantages of the present invention in which the following abbreviations mean:
amu, average mass unit; BMGY, rich standard medium containing glycerol; BMMY, rich standard medium containing methanol; ELISA, enzyme-linked immunosorbent assay; G418, geneticin; HBV, hepatitis B virus; HBcAg, HBV core antigen; HBeAg, HBV e antigen; HBsAg, HBV surface antigen; anti-HBc, antibody to HBcAg; MALDI-TOF, matrix-assisted laser-desorption ionisation-time-of-flight; Mut+, wild-type methanol utilization; Muts, slower methanol utilization.